I (the PI) have an extremely strong background in human and mouse molecular genetics, winning numerous awards and prestigious fellowships for my postdoctoral training. I am now seeking to combine this with a strong interest in reproduction and development. My long-term aims are to become an established investigator in the field of reproductive genetics and focus into the biology of folliculogenesis. This research proposal is based on a paradigm that has been successfully employed in genetic analysis for many years: the approach of studying patients falling into the extremes of a normal distribution for a biological trait (e.g., blood pressure) has been particularly successful in identifying genes for multifactorial disorders (e.g., hypertension). We propose to employ a similar strategy and perform a mutation screen in women that produce low or high numbers of oocytes after stimulation with FSH. We will examine the FSH receptor (FSHR) and its signaling cascades that promote follicular development. Common allelic variants of the FSHR have been implicated with variable ovarian response during stimulation. We hypothesize that additional mutations/polymorphisms in the FSHR gene resulting in altered structure and function of the FSHR may cause low or high response to FSH in women undergoing ovarian stimulation. Previous studies of FSHR variants in women undergoing in vitro fertilization (IVF) were performed on genomic DNA, which can fall short of detecting substitutions in regulatory regions, such as 5' and 3' UTR and introns. In our own preliminary studies, we examined cDNA from cumulus cells collected from oocytes in women being treated for IVF, and we specifically targeted young patients (<36 years) in the lower 25% and higher 25% oocyte number response during stimulation. We have identified a high incidence of alternatively spliced products in this select population. In this proposal we, therefore, aim to: firstly, continue the mutation analysis of FSHR cDNA in women with aberrant response to FSH stimulation and to further explore whether there is a correlation between identified variants and the number of produced oocytes; and secondly, to express these variants in cell lines and examine their ability to initiate downstream signaling cascades when stimulated with FSH. The in vitro system for functional analysis will be helpful in trying new combinations of drugs and for our future studies with the goal of discovering the factors that guide successful folliculogenesis. [unreadable] [unreadable] [unreadable]